Prior Art
Immunological methods for quantitative determinations based on the use of non-radioactive markers have been developed during the latest decade. One of the most important improvements within this research was the development of homogeneous immunoanalytical systems, which Rubinstein and his collaborators (see: (1972) Biochem. Biophys. Res. Com. 47, 846-851) defined as determinations which do not require any separation step. As the separation step can be left out, one source of errors is eliminated and at the same time, the execution of the analysis becomes easier, which flavors the automatization of an analysis method.
A homogeneous immunoanalysis thus means a system where both the reaction between antibody and ligand (hapten and antigen) and the determination of the degree of reaction of these are carried out in a homogeneous solution. The separation between "free" and "antibody-bound" compound can be avoided if the properties of the marker are affected by the reaction between antibody and ligand. In principle, two different types of homogeneous immunoassay systems have been developed. The reaction affects the physical properties of the marker or the reaction affects via the marker a biological activity which can be followed. In most homogeneous immunoassay systems a labelled ligand (Lg-M) is used, which reacts with a specific antibody (Ab) and an unknown ligand (Lg) the amount of which is to be determined. ##STR1##
When the antibody binds the labelled ligand the signal used for the analysis will be modified. In such a system the intensity of the measured signal can directly be related to the unknown concentration of the ligand in the sample. Immunochemical markers, which have been used to label ligands and which after the reaction with the antibody due to a changed physical environment give a modified signal, comprise free radicals (see Leute et al. (1972) JAMA 221, 1231-1234), fluorescent molecules (see Ullman et al. (1976) J. Biol. Chem. 27, 4172-4178) and chemiluminescent molecules (see Kohen et al. (1979) Febs Letters 104, 201-205).
A relatively large number of homogeneous fluorescence spectroscopic immunoanalytical methods have been developed, all of which are based on the fact that the biospecific affinity reaction affects in one way or the other the physical properties of the marker so that a change of signal can be registered. This change of signal which is fluorescence spectroscopically registered can for example consist of quenching, change in polarization, excitation transfer or protection. Comprehensive surveys have recently been published which give a good survey of the fluoroimmunoassay determinations known at present which can be carried out in homogeneous systems (see Smith et al. (1981) Ann. Clin. Biochem. 18, 253-274, Ullman (1981) "Recent Advances in Fluorescence Immunoassay Techniques".
In the above mentioned surveys the limitations to which both heterogeneous and homogeneous fluorescence immunoassay determinations are subject are also described, namely the high background fluorescence which follows all conventional fluorescent markers and fluorescence spectroscopic analyses. This has constituted the greatest limitation for a further development of sensitive methods of analysis based on fluorescence.